摘要
目的本实验拟观察不同氧浓度下破骨细胞诱导过程中的分化发育,寻找破骨细胞体外培养的适宜氧浓度,为骨转换平衡的修复提供依据。方法取野生型C57B/L小鼠(2个月龄左右,雄性)骨髓进行破骨细胞的诱导培养。用RANKL(10ng/ml)和M-CSF(10ng/ml)联合的诱导方案,将小鼠骨髓中单核-巨噬细胞系体外诱导为破骨细胞样细胞。将原代破骨细胞置于20%O2、7%O2、2%O2下诱导培养,MOCP5不同氧浓度下普通培养。用MTT法检测MOCP5的增殖变化,用抗酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞形成的变化,并进行TRAP阳性细胞计数,用象牙骨片骨吸收陷窝甲苯胺蓝染色检测破骨细胞骨吸收活性的变化。结果骨髓中单核-巨噬细胞体外经RANKL和M-CSF联合诱导可分化为多核的破骨细胞样细胞,在诱导第3天细胞开始融合,第5天TRAP染色强阳性,第8天可见象牙骨片上形成圆形、椭圆形、腊肠形等多种形态的骨吸收陷窝。MTT检测显示MOCP5在20%O2培养时一直处于增殖状态,7%O2条件下由增殖期进入平台期,2%O2时增殖缓慢且没有规律。20%O2、7%O2、2%O2培养下形成的TRAP阳性破骨细胞数分别为22±5.97、34±2.97、7±1.39(P<0.05),原代诱导的破骨细胞在20%O2、7%O2、2%O2形成的骨吸收陷窝面积(μm2)分别为3892.28±1642.78、5356.7±1655.6、2573±994.48(P<0.05)。结论体外RANKL和M-CSF联合可将骨髓单核-巨噬细胞诱导成多核的破骨细胞样细胞作为破骨细胞的研究模型。常氧条件下破骨细胞的TRAP阳性细胞数和骨吸收活性均低于7%O2。7%O2培养下的破骨细胞更接近于体内生理状态的破骨细胞。
Objective To examine the effect of oxygen tension on development, differentiation and function of osteoclasts in vitro and hope to find the most appropriate oxygen tension for osteoclasts culture. To provide the experiment foundation for the balance restoring of bone transformation. Methods Long bones were dissected from 8-week-old C57B/L mice. The femur and tibiae were cut across the epiphyses and the marrow was flushed out. The cell suspension and MOCP5 （ preosteoclast ） were cultured in different oxygen tension （ 20 % O2,7 % O2 , 2 % O2 ） respectively, 10 ng/ml M-CSF and 10 ng/ml sRANKL were added during the culture process. MTT assay was used to measure MOCP5 proliferation. Cells were stained for tartarate-resistant acid phosphatase （TRAP） and the total number of TRAP-positive muhinucleated osteoclasts was quantified. Ivory discs were stained with Toluidine bule to assess osteoclastic resorption. Results Monocyte/maerophage in bone marrow can be induced into muhinuclear cells incubated with M-CSF and RANKL in vitro. Cell fusion appeared on the 3rd day. TRAP-positive multinuclear cells were formed on the 5th day. Rounded, elliptic and sausage-liked bone lacuna was seen on the 8th day. MOCP5 kept proliferating in common oxygen tension, but it showed natural cell growth curve in 7 % O2, and it proliferated slowly in 2% O2. TRAP-positive muhinuclear cells were 22 ± 5.97, 34 ± 2.97, 7 ± 1.39（ P 〈 0.05） respectively in 20% O2 , 7% O2 , 2% O2. The area of bone lacuna were 3892.28 ± 1642.78μm^2 , 5356.7 ± 1655.69μm^2 , 2573 ± 994.48μm^2 （ P 〈 0.05 ） respectively at 20 % O2 , 7 % O2, 2 % O2 . Conclusion Monocyte/ macrophage derived from bone marrow can be induced into multinuclear cells incubated with M-CSF and RANKL in vitro. TRAP-positive multinuclear cells and bone lacuna area at common oxygen tension are lesser than at 7 % O2 . The 7% O2 was in favor of osteoclasts development, differentiation and function.
出处
《中国骨质疏松杂志》
CAS
CSCD
2008年第5期313-317,共5页
Chinese Journal of Osteoporosis
基金
上海市教委科研基金项目(06BZ033)
